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Endosperm Culture of Coconut a Dissertation ENDOSPERM CULTURE OF COCONUT A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE MAY 1996 By Lazarus Agus Sukamto Dissertation Committee: Yoneo Sagawa, Chairperson John T. Kunisaki Kenneth Y. Takeda David T. Webb James A. Silva 11 We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Horticulture. DISSERTATION COMMITTEE _ A 0,0xu'Z^ / Chairpej?^on Ill ACKNOWLEDGEMENT S Sincere thanks to OTO-BAPPENAS through MUCIA for funding my study, my advisor Dr. Yoneo Sagawa for continuous advice and support. I wish to thank my dissertation committee members, Drs. David Webb, James Silva, Kenneth Takeda and Mr. John Kunisaki who gave me support, critical advice and sugges­ tions. I also wish to thank Dr. Osamu Kawabata for statisti­ cal help and Dr. Se-Young Kim and other persons for help. Finally, I am very grateful to my wife Catharina for under­ standing and caring, my son Leo and my daughter Lei for joy. IV ABSTRACT The growth and differentiation of endosperm tissue of coconut cv. Samoan Dwarf in vitro were described. Endosperm tissues produced callus profusely without any enclosing embryo. Explants formed callus three weeks after culture. Callogenesis occurred in over 95% of all treatments. There was no significant difference in callogenesis between fruit sources, antipodal and micropylar tissues, 2,4- dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6 - trichloropicolinic acid (picloram) as well with 6 - benzylaminopurine (BAP). Auxin and cytokinin were not necessary to initiate callogenesis. Growth rate was influenced by fruit source and concentration of 2,4-D and picloram but not by endosperm region of fruit, two types of auxin (2,4-D and picloram) and addition of BAP. Growth rate of tissues increased substantially between 9 to 25 weeks but decreased by 31 weeks of culture. 2,4-D and picloram (lO'^M) inhibited growth rate in the beginning. The growth rate of the control was greater than other treatments at 31 weeks after culture. Tissue (endosperm and callus) browning occurred but did not inhibit tissue growth. In growth, tissue color changed from yellowish white, brown to black from which new yellowish white callus was produced. Callus structure changed from compact to friable after several transfers. V Morphogenesis occurred in endosperm callus of antipodal tissue initially treated with lO'^M picloram after 21 weeks of culture. This "organ" was elongate, opaque and grew slowly. Its shape changed from triangular with several lumps on the surface to cylindrical after 14 months. Morphogenesis also occurred on endosperm callus treated with 207.04xl0'^M picloram after 17 months. Histological study of endosperm callus showed structures which resembled proembryos, embryos with suspensors, promeristemoids and meristemoids. While "organ­ like" structure showed a meristematic layer with a dermal layer, cortex-like region and central vascular tissue; there were many small protuberances which resembled embryoids and shoot with tunica and corpus. Callogenesis and morphogenesis (shoot organogenesis) occurred in coconut endosperm in vitro. VI TABLE OF CONTENTS Acknowledgements.............................................. iii Abstract.........................................................iv List of Tables................................................ vii List of Figures.................................................ix List of Abbreviations....................................... xiii Chapter I. Introduction.........................................1 Chapter II. Literature review................................... 3 Endosperm culture.........................................10 Factors for culture of coconut endosperm...............12 1. Maturity of explant............................ 12 2. Presence or absence of the zygotic embryo....14 3. Culture medium.................................. 22 4. Growth regulators...............................24 5. Browning......................................... 32 6 . Culture period.................................. 35 Chapter III. Initiation of callogenesis.................... 37 Introduction.............................................. 37 Materials and methods.................................... 38 Results and discussion................................... 41 Chapter IV. Callus growth and morphogenesis................ 64 Introduction.............................................. 64 Materials and methods.................................... 65 Results and discussion................................... 66 Chapter V. Histological study.................................88 Introduction.............................................. 88 Materials and methods.................................... 90 Results and discussion................................... 92 Chapter VI. General discussion.............................. 108 Appendix....................................................... 115 References..................................................... 139 Vll LIST OF TABLES Table page 1. Coconut cultures reported from various tissues..........5 2. Endosperm cultures reported from various plants....... 15 3. Analysis for percentage of callogenesis after 31 weeks of culture....................................... 115 4. A summary of the percentage of callogenesis after 31 weeks of culture....................................... 116 5. Analysis for average tissue browning on the duration of culture...................................... 117 6 . A summary of browning score of tissues on the duration of culture....................................... 121 7. Analysis for effect of transfer on growth rate ........ 122 8 . Analysis of growth rate after 9 weeks of culture...... 123 9. Analysis for effect of fruit source on growth rate after 9 weeks of culture............................ 124 10. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 9 weeks of culture.................................................... 125 11. Analysis of growth rate after 16 weeks of culture.... 126 12. Analysis for effect of fruit source on growth rate after 16 weeks of culture.......................... 127 13. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 16 weeks of culture.................................................... 128 14. Analysis of growth rate after 21 weeks of culture.... 129 15. Analysis for effect of fruit source on growth rate after 21 weeks of culture.......................... 130 16. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 21 weeks of culture.................................................... 131 17. Analysis of growth rate after 26 weeks of culture.... 132 18. Analysis for effect of fruit source on growth rate after 26 weeks of culture.......................... 133 Vlll LIST OF TABLES (continued) Table page 19. Analysis for effect of 2,4-D and picloraiti concentrations on growth rate after 26 weeks of culture.................................................... 134 20. Analysis of growth rate after 31 weeks of culture.... 135 21. Analysis for effect of fruit source on growth rate after 31 weeks of culture.......................... 136 22. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 31 weeks of culture.................................................... 137 23. A summary of growth rate (g per week) of tissues on the duration of culture.............................. 138 IX LIST OF FIGURES Figure page 1. Varied growth and development of endosperm after 4 weeks in control in control. Endosperm tissue was still white, other was brownish and swollen, while others turned dark brown and formed callus.............. 52 2. Callus first appeared on the top of the explant, another on the side of the explant which touched the medium after 7 weeks in control.......................... 52 3. Callus appearances: pale color and slightly friable after 15 months of tissues treated with lO'^M 2,4-D and another with addition of lO'^M BAP ...................53 4. Tissue (endosperm and callus) browning were varied from little (I = score 1 ) to medium (III = score 2) and high (III = score 3) in control after 21 weeks....53 5. Varied colors of callus and new yellowish white callus grown on the black callus of antipodal tissue treated with 2,4-D and lO'^M BAP after 13 months............................................ 54 6 . Average browning score of all treatments............... 55 7. Effect of endosperm region on tissue browning......... 56 8 . Effect of 2,4-D and picloram on tissue browning....... 57 9. Effect of 2,4-D and picloram concentrations on tissue browning after 9 weeks of culture............... 58 10. Effect of 2,4-D and picloram concentrations on tissue browning after 16 weeks of culture.............. 59 11. Effect of 2,4-D and picloram concentrations on tissue browning after 21 weeks of culture.............. 60 12. Effect of 2,4-D and picloram concentrations on tissue browning after 2 6 weeks of culture.............. 61 13. Effect of 2,4-D and picloram concentrations on tissue browning after 31 weeks of culture.............. 62 14. Effect of lO'^M BAP on tissue browning.................. 63 15. Average tissue growth of all treatments................ 74 16. Average growth rate of all treatments.................. 75 X LIST OD FIGURES (continued) Figure page 17. Growth
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